MAB1675 Sigma-AldrichAnti-Cytokeratin 19 Antibody, clone E6

BACKGROUND AND AIMS: Activation of the oval cell compartment occurs in the liver when hepatocytes are functionally compromised and/or unable to divide. Our goal was to investigate the systemic signals responsible for determining the efficiency of oval cell-mediated liver regeneration, focusing on the Notch signaling cascade. METHODS: The established oval cell induction protocol of 2-acetylaminofluorine (2-AAF) implantation followed by 70% surgical resection of the liver (partial hepatectomy, PH) was employed in a rat model. This oval cell induction model was further combined with injections of a γ-secretase inhibitor (GSI XX) to examine the effects of Notch inhibition on oval cell-aided regeneration of the liver. RESULTS: Notch signaling was found to be upregulated at the peak of oval cell induction during 2AAF-PH alone. Treatment with GSI XX led to interruption of the Notch signal, as shown by a decrease in expression of Hes1. While there was a robust oval cell response seen at day 11 post-PH, there was a measurable delay in differentiation when Notch was inhibited. This was confirmed morphologically as well as by immunohistochemistry for the oval cell markers, α-fetoprotein, OV-6, and CK19. The hepatocytes seen at day 22 demonstrated an enhanced hepatocellular mitoinhibition index (p21(Waf1)/Ki67), suggestive of dysregulated proliferation and cell cycle progression. Moreover, these hepatocytes exhibited decreased expression of hepatocyte functional markers, such as cytochrome P450 and glucose-6-phosphatase-α. CONCLUSIONS: Taken together, these results identify the Notch signaling pathway as a potent regulator of differentiation and proliferation in oval cells, which is necessary for functional for repair of the liver by oval cells.

Very little remains known about the regulation of human organ stem cells (in general, and during the aging process), and most previous data were collected in short-lived rodents. We examined whether stem cell aging in rodents could be extrapolated to genetically and environmentally variable humans. Our findings establish key evolutionarily conserved mechanisms of human stem cell aging. We find that satellite cells are maintained in aged human skeletal muscle, but fail to activate in response to muscle attrition, due to diminished activation of Notch compounded by elevated transforming growth factor beta (TGF-beta)/phospho Smad3 (pSmad3). Furthermore, this work reveals that mitogen-activated protein kinase (MAPK)/phosphate extracellular signal-regulated kinase (pERK) signalling declines in human muscle with age, and is important for activating Notch in human muscle stem cells. This molecular understanding, combined with data that human satellite cells remain intrinsically young, introduced novel therapeutic targets. Indeed, activation of MAPK/Notch restored 'youthful' myogenic responses to satellite cells from 70-year-old humans, rendering them similar to cells from 20-year-old humans. These findings strongly suggest that aging of human muscle maintenance and repair can be reversed by 'youthful' calibration of specific molecular pathways.

The paired box domain gene Pax7 plays a pivotal role in satellite cell physiology and may represent one of the candidate genes influencing the dynamic stages of early post-natal growth observed in pig. Quiescent satellite cells express Pax7 and, when activated, they co-express the myogenic bHLH protein MyoD. The aims of this study were to investigate, by immunohistochemistry, the putative differential expression of Pax7 and to ascertain the amount of activated satellite cells (Pax7(+)/MyoD(+)) in myogenic cells isolated at different post-natal time points and in adults. Our results indicate that Pax7(+) cells represent between 10 and 15% of the whole myogenic cell population found at birth indicating that these cells provide a modest contribution to the development of new fibres. The number of activated satellite cells (Pax7(+)/MyoD(+)) was scarce after birth but it was higher respect to adults. An interesting result was that at 1 month after birth the number of Pax7(+) cells had increased within the pool of myogenic cells with respect to myogenic cells extracted at birth. We speculate that Pax7 might be one of the molecules involved in controlling the proliferation/differentiation ratio in the pool of satellite cells present in post-natal porcine skeletal muscles.

We established genetically engineered ES (ZHTc6-MyoD) cells that harbor a tetracycline-regulated expression vector encoding myogenic transcriptional factor MyoD, for the therapy of muscle diseases, especially Duchenne muscular dystrophy (DMD). Almost all the ZHTc6-MyoD cells were induced into muscle lineage after removal of tetracycline. The undifferentiated ZHTc6-MyoD cells are Sca-1+ and c-kit+, but CD34-, all well-known markers for mouse hematopoietic stem cells. In addition, they are able to maintain themselves in the undifferentiated state, even after one month of culture. Therefore, it is possible to obtain a large quantity of ZHTc6-MyoD cells in the undifferentiated state that maintain the potential to differentiate only into muscle lineage. Additionally, at two weeks post-injection of these cells into muscle of mdx, a model mouse of DMD, clusters of dystrophin-positive myofibers were observed at the injection site. Therefore, ES cells have considerable therapeutic potential for treating muscle diseases.

Cystathionine gamma-lyase (CSE) is an enzyme catalyzing cystathionine and cysteine to yield cysteine and hydrogen sulfide (H(2)S), respectively. This study aimed to examine if H(2)S generated from the enzyme could serve as an endogenous regulator of hepatobiliary function. Gas chromatographic analyses indicated that, among rat organs herein examined, liver constituted one of the greatest components of H(2)S generation in the body, at 100 mumol/g of tissue, comparable to that in kidney and 1.5-fold greater than that in brain, where roles of the gas in the regulation of neurotransmission were reported previously. At least half of the gas amount in the liver appeared to be derived from CSE, because blockade of the enzyme by propargylglycine suppressed it by 50%. Immunohistochemistry revealed that CSE occurs not only in hepatocytes, but also in bile duct. In livers in vivo, as well as in those perfused ex vivo, treatment with the CSE inhibitor induced choleresis by stimulating the basal excretion of bicarbonate in bile samples. Transportal supplementation of NaHS at 30 mumol/L, but not that of N-acetylcysteine as a cysteine donor, abolished these changes elicited by the CSE inhibitor in the perfused liver. The changes elicited by the CSE blockade did not coincide with alterations in hepatic vascular resistance, showing little involvement of vasodilatory effects of the gas in these events, if any. These results first provided evidence that H(2)S generated through CSE modulates biliary bicarbonate excretion and is thus a determinant of bile salt-independent bile formation in the rat liver.

Intercalated epithelial cells exist in a spectrum of phenotypes; at one extreme, beta cells secrete HCO3 by an apical Cl/HCO3 exchanger and a basolateral H+ ATPase. When an immortalized beta cell line is seeded at high density it deposits in its extracellular matrix (ECM) a new protein, hensin, which can reverse the polarity of several proteins including the Cl/HCO3 exchanger (an alternately spliced form of band 3) and the proton translocating ATPase. When seeded at low density and allowed to form monolayers these polarized epithelial cells maintain the original distribution of these two proteins. Although these cells synthesize and secrete hensin, it is not retained in the ECM, but rather, hensin is present in a large number of intracellular vesicles. The apical cytoplasm of low density cells is devoid of actin, villin, and cytokeratin19. Scanning electron microscopy shows that these cells have sparse microvilli, whereas high density cells have exuberant apical surface infolding and microvilli. The apical cytoplasm of high density cells contains high levels of actin, cytokeratin19, and villin. The cell shape of these two phenotypes is different with high density cells being tall with a small cross-sectional area, whereas low density cells are low and flat. This columnarization and the remodeling of the apical cytoplasm is hensin-dependent; it can be induced by seeding low density cells on filters conditioned by high density cells and prevented by an antibody to hensin. The changes in cell shape and apical cytoskeleton are reminiscent of the processes that occur in terminal differentiation of the intestine and other epithelia. Hensin is highly expressed in the intestine and prostate (two organs where there is a continuous process of differentiation). The expression of hensin in the less differentiated crypt cells of the intestine and the basal cells of the prostate is similar to that of low density cells; i.e., abundant intracellular vesicles but no localization in the ECM. On the other hand, as in high density cells hensin is located exclusively in the ECM of the terminally differentiated absorptive villus cells and the prostatic luminal cell. These studies suggest that hensin is a critical new molecule in the terminal differentiation of intercalated cell and perhaps other epithelial cells.

In a previous publication (Rodriguez, M.L., M. Brignoni, and P.J.I. Salas. 1994. J. Cell Sci. 107: 3145-3151), we described the existence of a terminal web-like structure in nonbrush border cells, which comprises a specifically apical cytokeratin, presumably cytokeratin 19. In the present study we confirmed the apical distribution of cytokeratin 19 and expanded that observation to other epithelial cells in tissue culture and in vivo. In tissue culture, subconfluent cell stocks under continuous treatment with two different 21-mer phosphorothioate oligodeoxy nucleotides that targeted cytokeratin 19 mRNA enabled us to obtain confluent monolayers with a partial (40-70%) and transitory reduction in this protein. The expression of other cytoskeletal proteins was undisturbed. This downregulation of cytokeratin 19 resulted in (a) decrease in the number of microvilli; (b) disorganization of the apical (but not lateral or basal) filamentous actin and abnormal apical microtubules; and (c) depletion or redistribution of apical membrane proteins as determined by differential apical-basolateral biotinylation. In fact, a subset of detergent-insoluble proteins was not expressed on the cell surface in cells with lower levels of cytokeratin 19. Apical proteins purified in the detergent phase of Triton X-114 (typically integral membrane proteins) and those differentially extracted in Triton X-100 at 37 degrees C or in n-octyl-beta-D-glycoside at 4 degrees C (representative of GPI-anchored proteins), appeared partially redistributed to the basolateral domain. A transmembrane apical protein, sucrase isomaltase, was found mispolarized in a subpopulation of the cells treated with antisense oligonucleotides, while the basolateral polarity of Na+-K+ATPase was not affected. Both sucrase isomaltase and alkaline phosphatase (a GPI-anchored protein) appeared partially depolarized in A19 treated CACO-2 monolayers as determined by differential biotinylation, affinity purification, and immunoblot. These results suggest that an apical submembrane cytoskeleton of intermediate filaments is expressed in a number of epithelia, including those without a brush border, although it may not be universal. In addition, these data indicate that this structure is involved in the organization of the apical region of the cytoplasm and the apical membrane.